Conformationally Constrained Isoquinolinones as Orally Efficacious Hepatitis B Capsid Assembly Modulators.

Isoquinolinone-based HBV capsid assembly modulators that bind at the dimer:dimer interface of HBV core protein have been shown to suppress viral replication in chronic hepatitis B patients. Analysis of their binding mode by protein X-ray crystallography has identified a region of the small molecule where the application of a constraint can lock the preferred binding conformation and has allowed for further optimization of this class of compounds. Key analogues demonstrated single digit nM EC50 values in reducing HBV DNA in a HepDE19 cellular assay in addition to favorable ADME and pharmacokinetic properties, leading to a high degree of oral efficacy in a relevant in vivo hydrodynamic injection mouse model of HBV infection, with 12e effecting a 3 log10 decline in serum HBV DNA levels at a once daily dose of 1 mg/kg. Additionally, maintenance of activity was observed in clinically relevant HBV core protein variants T33N and I105T.

Rational Design, Synthesis, and Structure-Activity Relationship of a Novel Isoquinolinone-Based Series of HBV Capsid Assembly Modulators Leading to the Identification of Clinical Candidate AB-836.

Inhibition of Hepatitis B Virus (HBV) replication by small molecules that modulate capsid assembly and the encapsidation of pgRNA and viral polymerase by HBV core protein is a clinically validated approach toward the development of new antivirals. Through definition of a minimal pharmacophore, a series of isoquinolinone-based capsid assembly modulators (CAMs) was identified. Structural biology analysis revealed that lead molecules possess a unique binding mode, exploiting electrostatic interactions with accessible phenylalanine and tyrosine residues. Key analogs demonstrated excellent primary potency, absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic properties, and efficacy in a mouse model of HBV. The optimized lead also displayed potent inhibition of capsid uncoating in HBV-infected HepG2 cells expressing the sodium-taurocholate cotransporting polypeptide (NTCP) receptor, affecting the generation of HBsAg and cccDNA establishment. Based on these results, isoquinolinone derivative AB-836 was advanced into clinical development. In Phase 1b trials, AB-836 demonstrated >3 log10 reduction in serum HBV DNA, however, further development was discontinued due to the observation of incidental alanine aminotransferase (ALT) elevations.

Discovery of C-Linked Nucleoside Analogues with Antiviral Activity against SARS-CoV-2.

The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n, which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.

Discovery of Clinical Candidate CEP-37440, a Selective Inhibitor of Focal Adhesion Kinase (FAK) and Anaplastic Lymphoma Kinase (ALK).

Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

Piperidine-3,4-diol and piperidine-3-ol derivatives of pyrrolo[2,1-f][1,2,4]triazine as inhibitors of anaplastic lymphoma kinase.

The diastereoselective synthesis and biological activity of piperidine-3,4-diol and piperidine-3-ol-derived pyrrolotriazine inhibitors of anaplastic lymphoma kinase (ALK) are described. Although piperidine-3,4-diol and piperidine-3-ol derivatives showed comparable in vitro ALK activity, the latter subset of inhibitors demonstrated improved physiochemical and pharmacokinetic properties. Furthermore, the stereochemistry of the C3 and C4 centers had a marked impact on the in vivo inhibition of ALK autophosphorylation. Thus, trans-4-aryl-piperidine-3-ols (22) were more potent than the cis diastereomers (20).

Anaplastic lymphoma kinase inhibitors as anticancer therapeutics: a patent review.

INTRODUCTION: Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase from the insulin receptor superfamily, is implicated in the oncogenesis of numerous cancers including anaplastic large-cell lymphoma, non-small-cell lung cancer, diffuse large B-cell lymphoma, inflammatory myofibroblastic tumors, glioblastoma, as well as neuroblastoma. The root cause for these specific cancers has been identified as aberrant ALK kinase activity, which has been shown to be associated with specific gene translocations, single-point mutations, gene amplification and/or overexpression. The direct inhibition of ALK with small-molecule inhibitors represents a viable therapeutic intervention that has achieved clinical proof of concept. AREAS COVERED: Small-molecule ALK inhibitors covered in the patent literature from 2010 to September 2013 are described. Relevant peer-reviewed journal articles that describe discovery and development of the above-identified ALK inhibitors are also discussed. Keyword-based (e.g., ALK, anaplastic lymphoma kinase) literature searches were conducted in Scifinder®. EXPERT OPINION: Novel ALK inhibitors continued to be discovered at a fast pace over the covered period, with many distinct chemotypes emerging. Crizotinib received FDA approval in 2011, and six additional ALK inhibitors have entered clinical trials. The focus of ALK research appears to have shifted toward inhibitors that display activity against resistant mutants unearthed in clinical studies with crizotinib.

A selective, orally bioavailable 1,2,4-triazolo[1,5-a]pyridine-based inhibitor of Janus kinase 2 for use in anticancer therapy: discovery of CEP-33779.

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

Strategies to mitigate the bioactivation of 2-anilino-7-aryl-pyrrolo[2,1-f][1,2,4]triazines: identification of orally bioavailable, efficacious ALK inhibitors.

Chemical strategies to mitigate cytochrome P450-mediated bioactivation of novel 2,7-disubstituted pyrrolo[2,1-f][1,2,4]triazine ALK inhibitors are described along with synthesis and biological activity. Piperidine-derived analogues showing minimal microsomal reactive metabolite formation were discovered. Potent, selective, and metabolically stable ALK inhibitors from this class were identified, and an orally bioavailable compound (32) with antitumor efficacy in ALK-driven xenografts in mouse models was extensively characterized.

2,7-Disubstituted-pyrrolotriazine kinase inhibitors with an unusually high degree of reactive metabolite formation.

There are numerous published studies establishing a link between reactive metabolite formation and toxicity of various drugs. Although the correlation between idiosyncratic reactions and reactive metabolite formation is not 1:1, the association between the two is such that many pharmaceutical companies now monitor for reactive metabolites as a standard part of drug candidate testing and selection. The most common method involves in vitro human microsomal incubations in the presence of a thiol trapping agent, such as glutathione (GSH), followed by LC/MS analysis. In this study, we describe several 2,7-disubstituted-pyrrolotriazine analogues that are extremely potent reactive metabolite precursors. Utilizing a UPLC/UV/MS method, unprecedented levels of GSH adducts were measured that are 5-10 times higher than previously reported for high reactive metabolite-forming compounds such as clozapine and troglitazone.

2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazines: new variant of an old template and application to the discovery of anaplastic lymphoma kinase (ALK) inhibitors with in vivo antitumor activity.

A novel 2,7-disubstituted-pyrrolo[2,1-f][1,2,4]triazine scaffold has been designed as a new kinase inhibitor platform mimicking the bioactive conformation of the well-known diaminopyrimidine motif. The design, synthesis, and validation of this new pyrrolo[2,1-f][1,2,4]triazine scaffold will be described for inhibitors of anaplastic lymphoma kinase (ALK). Importantly, incorporation of appropriate potency and selectivity determinants has led to the discovery of several advanced leads that were orally efficacious in animal models of anaplastic large cell lymphoma (ALCL). A lead inhibitor (30) displaying superior efficacy was identified and in depth in vitro/in vivo characterization will be presented.

Methanesulfonamido-cyclohexylamine derivatives of 2,4-diaminopyrimidine as potent ALK inhibitors.

The incorporation of R,R-1,2-diaminocyclohexane at C4 in a series of 2,4-diaminopyrimidines led to a number of ALK inhibitors in which optimized activity was achieved by conversion of the 2-amino group into a methanesulfonamide. Tumor growth inhibition was observed when an orally bioavailable analog was evaluated in a Karpas-299 tumor xenograft mouse model.

Novel 2,3,4,5-tetrahydro-benzo[d]azepine derivatives of 2,4-diaminopyrimidine, selective and orally bioavailable ALK inhibitors with antitumor efficacy in ALCL mouse models.

The synthesis and biological evaluation of potent and selective anaplastic lymphoma kinase (ALK) inhibitors from a novel class of 2,4-diaminopyrimidines, incorporating 2,3,4,5-tetrahydro-benzo[d]azepine fragments, is described. An orally bioavailable analogue (18) that displayed antitumor efficacy in ALCL xenograft models in mice was identified and extensively profiled.

Design, synthesis, and structural analysis of D,L-mixed polypyrrolinones. 2. Macrocyclic hexapyrrolinones.

The design, synthesis, and structural analysis of two macrocyclic D,L-alternating hexapyrrolinones have been achieved. These cyclic peptide mimics adopt a flat, hexagonal conformation, stabilized by intramolecular hydrogen bonding between adjacent pyrrolinone rings. Extensive NMR studies and X-ray analysis reveal, respectively, that the macrocyclic hexapyrrolinones aggregate in solution and in the solid state form staggered stacked nanotube-like assemblies.

Evolution of a total synthesis of (-)-kendomycin exploiting a Petasis-Ferrier rearrangement/ring-closing olefin metathesis strategy.

A convergent stereocontrolled total synthesis of (-)-kendomycin (1) has been achieved. The synthesis proceeds with a longest linear sequence of 21 steps, beginning with commercially available 2,4-dimethoxy-3-methylbenzaldehyde (12). Highlights of the synthesis include an effective Petasis-Ferrier union/rearrangement tactic to construct the sterically encumbered tetrahydropyran ring, a ring-closing metathesis to generate the C(4a-13-20a) macrocycle, an effective epoxidation/deoxygenation sequence to isomerize the C(13,14) olefin, and a biomimetic quinone-methide-lactol assembly to complete the synthesis.

Total synthesis of (-)-kendomycin exploiting a Petasis-Ferrier rearrangement/ring-closing olefin metathesis synthetic strategy.

The total synthesis of (-)-kendomycin (1), a novel macrocyclic polyketide with antibacterial and antitumor activity, was achieved in 21 steps (longest linear sequence) exploiting an effective Petasis-Ferrier union/rearrangement tactic to construct the tetrahydropyran ring, a ring-closing metathesis to generate the macrocycle, and a biomimetic quinone-methide-lactol assembly.

Design, synthesis, and binding affinities of pyrrolinone-based somatostatin mimetics.

[structure: see text] Tetrapyrrolinone somatostatin (SRIF) mimetics (cf. 1), based on a heterochiral (D,L-mixed) pyrrolinone scaffold, were designed, synthesized, and evaluated for biological activity. The iterative synthetic sequence, incorporating the requisite functionalized coded and noncoded amino acid side chains, comprised a longest linear synthetic sequence of 23 steps. Binding affinities at two somatostatin receptor subtypes (hsst 4 and 5) reveal micromolar activity, demonstrating that the d,l-mixed pyrrolinone scaffold can be employed to generate functional mimetics of peptide beta-turns.

Diastereoselective conjugate additions to pi-allylmolybdenum complexes: a stereocontrolled route to 3,4,5-trisubstituted gamma-butyrolactones.

[reaction: see text] pi-Allylmolybdenum complex 6b is obtained as a single isomer by Knoevenagel condensation of aldehyde 1 with Meldrum's acid. Conjugate additions of Grignard reagents to Meldrum's acid alkylidene derivative 6b are shown to be completely diastereoselective. Further functional group transformation of the 1,4-adducts, followed by demetalation, leads to trisubstituted tetrahydrofurans and gamma-butyrolactones. Whereas the synthesis of tetrahydrofurans (X = 2H) is not completely stereoselective, the gamma-butyrolactones (X = O) are obtained with good to excellent diastereoselectivities.